Changes in phospholipid composition and consequent loss of membrane integrity are correlated with loss of seed viability. Furthermore, phospholipid compositional changes affect the composition of the triacylglycerols (TAG), i.e. the storage lipids. Phospholipase D (PLD) catalyses the hydrolysis of phospholipids to phosphatidic acid, and PLDα is an abundant PLD isoform. Although wild-type (WT) seeds stored for 33 months were non-viable, 30%-50% of PLDα-knockdown (PLD-KD) soybean seeds stored for 33 months germinated. WT and PLD-KD seeds increased in lysophospholipid levels and in TAG fatty acid unsaturation during ageing, but the levels of lysophospholipids increased more in WT than in PLD-KD seeds. The loss of viability of WT seeds was correlated with alterations in ultrastructure, including detachment of the plasma membrane from the cell wall complex and disorganization of oil bodies. The data demonstrate that, during natural ageing, PLDα affects the soybean phospholipid profile and the TAG profile. Suppression of PLD activity in soybean seed has potential for improving seed quality during long-term storage.